The invention concerns methods for coating a substrate with at least one monolayer of self-assembling proteins as well as substrates obtainable thereby. The invention can be employed in chemical industry, biotechnology, sensor technology as well as in medical research.
Self-assembling proteins, like hydrophobins or surface layer proteins (S-layer proteins), have the tendency to undergo in aqueous solutions a quick and uncontrollable aggregate formation (the uncontrolled agglomeration of protein monomers to multimers of the self-assembling proteins) which complicates the preparation of homogeneous layers of the self-assembling proteins on a substrate. With regard to the invention, self-assembling proteins in the following are also simply referred to as “proteins”. Self-assembling proteins, by interactions of the protein molecules with each other, form a directed folded structure and association (molecular self-assemblage) and are suitable, by the formation of this extremely ordered structure, for very different applications in nanotechnology.
Generally, the Langmuir Blodgett method, the Langmuir Schäfer technique or the “layer-by-layer” process are used for building homogeneous layers of self-assembling proteins. In these methods, the substrate is submerged in a solution with protein monomers so that a layer of the protein monomers (monolayer) is deposited on the substrate. The layer-by-layer techniques are based exclusively on the utilization of physisorption of the protein monomers on the substrate surface. Hence, it is necessary to use high purity protein monomers. Because self-assembling proteins have a strong tendency to form aggregates, a long-term storage and use of already prepared protein solutions is not possible. Furthermore, coating of hardly accessible surfaces of the substrate (for example, pores of a substrate) is limited because hardly accessible sites on the substrate, such as for example pores, may become closed off by protein monomers that aggregate in uncontrolled fashion so that further monomers cannot penetrate into pores anymore.
DE 10 2005 051515 A1 discloses methods for coating surfaces with defined fusion hydrophobins. In this context, an aqueous solution having a pH value of ≥4 is contacted with a surface and the solvent is subsequently removed. Other additives, such as for example non-ionic surfactants and/or metal ions, may be added to the hydrophobin solution. The process permits however no controlled deposition of protein layers.
Moreover, processes are known for stabilizing solutions of monomers of self-assembling proteins. For example, a process is known where protein aggregates are dissolved by addition of trifluoro acetic acid (TFA) without damaging the proteins. In view of environmental regulations and the very corrosive properties of TFA, this method is suitable only to a very limited extent for industrial applications.
Furthermore, WO 01/57066 A2 discloses a process where the disulfide bonds within a hydrophobin are cleaved by addition of a strongly denaturing agent, like guanidine hydrochloride, so that the folded structure of the hydrophobin is dissolved. The renewed formation of disulfide bonds in the hydrophobin solution is avoided by addition of suitable agents for forming protective sulfhydryl groups so that the thus generated hydrophobin monomer-containing solutions are stable. For coating a substrate surface with self-assembling protein monomers, the removal of the protective group is then necessary which is effected by addition of an oxidizing agent.
WO 96/41882 A1 discloses hydrophobin assemblage at oil-water interphases. Furthermore, EP 1 252 516 A1 and WO 2005/068087 A1 disclose the immobilization of hydrophobins by means of thermal sample preparation or by shifting the pH value into the acidic range. However, both processes provide no information about the number and homogeneity of the generated hydrophobin layers, for which reason they cannot be used in an application in sensor technology or biomedicine.